Activation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in the pathogenesis of a variety of cancers. Recently, mutations in the gene encoding the p100α catalytic subunit of PI3K (PIK3CA) have been identified in several human cancers. The mutations primarily result in single amino acid substitutions, with >85% of the mutations in either exon 9 or 20. Multiple studies have shown that these mutations are observed in 18% to 40% of breast cancers. However, the phenotypic effects of these PIK3CA mutations have not been examined in breast epithelial cells. Herein, we examine the activity of the two most common variants, E545K and H1047R, in the MCF-10A immortalized breast epithelial cell line. Both variants display higher PI3K activity than wild-type p100α yet remain sensitive to pharmacologic PI3K inhibition. In addition, expression of p100α mutants in mammary epithelial cells induces multiple phenotypic alterations characteristic of breast tumor cells, including anchorage-independent proliferation in soft agar, growth factor-independent proliferation, and protection from anoikis. Expression of these mutant p110α isoforms also confers increased resistance to paclitaxel and induces abnormal mammary acinar morphogenesis in three-dimensional basement membrane cultures. Together, these data support the notion that the cancer-associated mutations in PIK3CA may significantly contribute to breast cancer pathogenesis and represent attractive targets for therapeutic inhibition.